Methods for the specific preparation of lysobactin fragments

ABSTRACT

The invention relates to methods for the targeted production of lysobactin derivatives by combined chemical and enzymatic modifications. In particular, the invention relates to method for preparing lysobactin fragment 4-11 by chemical reduction and cleavage of the resultant product by chymotrypsin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/800,102, filed May 4, 2007, now allowed, which is a continuation ofInternational Application No. PCT/EP2005/011364, filed Oct. 22, 2005,designating US, which claims priority from German Patent Application No.DE 10 2004 053 409.8, filed Nov. 5, 2004. The contents of theabove-referenced applications are incorporated herein by this referencein their entirety.

BACKGROUND OF THE INVENTION

The invention relates to methods for the targeted preparation oflysobactin derivatives by combined chemical and enzymatic modifications.In particular, the invention relates to a method for preparinglysobactin fragment 4-11 by chemical reduction and cleavage of theresultant product by chymotrypsin.

Lysobactin is a cyclic depsipeptide which originates from a screeningprogram for finding novel antibiotics acting in the biosynthesis ofbacterial cell walls (O'Sullivan J. et al., J. Antibiot. (1988)41(12):1740-1744 and Bonner, D. P. et al., J. Antibiot. (1988)41(12):1745-1751; Tymiak, A. A. et al., J. Org. Chem. (1989)54:1149-1157). It shows strong activity against Gram-positive aerobicand anaerobic bacteria. An unusual feature is the high number ofnon-proteinogenic amino acids in the molecule. In addition to the threeβ-hydroxyamino acids (2S,3R)-β-hydroxyleucine,(2S,3R)-β-hydroxyphenylalanine and (2S,3S)-(β-hydroxyasparagine, theD-amino acids D-leucine and D-arginine as well as allo-threonine alsooccur. This complexity and the size of the natural product lysobactinare a great hurdle for targeted chemical modifications.

SUMMARY OF THE INVENTION

It is therefore one object of the present invention to provide novel andalternative synthesis methods for the targeted synthesis of lysobactinfragments in order to make the preparation of novel antibiotics usinglysobactin fragments possible.

A solution is offered by targeted enzymatic cleavage, targeted enzymaticproduction and subsequent linkage of lysobactin fragments in combinationwith chemical modification steps, for example hydrogenation.

Enzymatic digestion experiments of lysobactin and the open-chain formobtained by hydrolysis (“open-chain lysobactin”; compound of formula(I)) with enzymes such as pepsin, trypsin, chymotrypsin and mucosalpeptidase showed no (such as in the case of pepsin, for example) or onlyinadequate enzymatic digestion (R. A. Blackburn et al., Drug Metab.Dispos. (1993) 21(4):573-579). Very slow inefficient enzymatic cleavageof lysobactin occurs only after the opening of the ring by hydrolysis inthe buffer used. This leads as an unwanted side reaction to side-chaindeamidation at the (2S,3S)-β-hydroxyasparagine. That is theβ-hydroxyasparagine unit is converted into a β-hydroxyaspartate unit.

Surprisingly it has been found that the lysobactin fragment 4-11 can beproduced highly efficiently and quantitatively by enzymatic cleavagewith chymotrypsin from dihydrolysobactin (compound of formula (II)) andoctahydrolysobactin (compound of formula (III)) as well as from amixture of both components. The cleavage takes place so rapidly that thefragments 1-3 and 4-11 are formed virtually after the combination of thereaction partners (substrate and enzyme). Unwanted side reactions in theamino acid side chains do not take place.

Dihydrolysobactin and octahydrolysobactin are obtained by hydrogenolyticopening of lysobactin with hydrogen, whereby the(2S,3R)-3-hydroxyphenylalanine unit is converted into a phenylalanine or3-cyclohexylalanine unit. The resulting lysobactin fragmentsdihydrolysobactin and octahydrolysobactin are then used for theenzymatic digestion.

Surprisingly, dihydrolysobactin and octahydrolysobactin are also goodsubstrates for other enzymes, so that other fragments can also beproduced in high yield by selection of the enzyme.

The invention relates to a method for preparing dihydrolysobactin and/oroctahydrolysobactin, in which lysobactin is converted todihydrolysobactin and/or ocatahydrolysobactin by hydrogenolytic ringopening with hydrogen in the presence of a hydrogenation catalyst in asolvent.

Hydrogenation catalysts are, for example, palladium, ruthenium, rhodium,iridium and platinum catalysts, or Raney nickel. These catalysts can beused as salts (for example platinum dioxide, rhodium(III) chloride) oras supported catalysts (for example palladium on carbon (5-30%) orrhodium on carbon (5%)). Suitable support materials for supportedcatalysts are, for example, activated carbon, kieselguhr, silica gel,bentonites, kaolin, pumice, aluminosilicates or aluminum oxide. Apreferred support material is activated carbon.

Bimetallic catalysts or else multicomponent catalysts can also be used.

Preference is given to palladium catalysts, for example palladium oncarbon (5-30%), particular preference is given to palladium on carbon(10%).

The hydrogenolytic ring opening generally takes place in a solvent,preferably in a temperature range from room temperature to 150° C.,preferably in a temperature range from room temperature to 80° C., in anatmospheric pressure range from atmospheric pressure to 200 bar,preferably in a pressure range from 3 to 80 bar.

Solvents are, for example, alcohols such as methanol, ethanol, orisopropanol, or mixtures of alcohols with water, or acetic acid oraqueous solutions of acetic acid, or THF-water mixtures, ordioxane-water mixtures, or else ternary mixtures of the abovementionedsolvents, for example isopropanol-water-acetic acid. Preference is givento an isopropanol-water mixture.

The invention further relates to a method for preparing lysobactinfragment 4-11 and lysobactin fragment 1-3, in which dihydrolysobactinand/or octahydrolysobactin are enzymatically cleaved to give lysobactinfragment 4-11 and lysobactin fragment 1-3.

Preference is given to an enzymatic cleavage of dihydrolysobactin and/oroctahydrolysobactin, whereby a eukaryotic serine protease or a microbialserine protease is used as enzyme.

Eukaryotic serine proteases are, for example, chymotrypsin, cathepsin G,chymase or other enzymes of the chymotrypsin family, or other eukaryoticserine proteases which cleave after aromatic amino acids, preference isgiven to chymotrypsin.

Microbial serine proteases are, for example, subtilisin, proteinase K,Streptomyces protease A or other enzymes which cleave after aromaticamino acids, preference is given to subtilisin.

The invention further relates to a method for the enzymatic cleavage ofdihydrolysobactin and/or octahydrolysobactin to give smaller lysobactinfragments.

The invention accordingly further relates to a method for preparinglysobactin fragment 3-11 and/or lysobactin fragment 5-11 and/orlysobactin fragment 4-10 and/or lysobactin fragment 1-9, characterizedin that dihydrolysobactin and/or octahydrolysobactin are enzymaticallycleaved to give lysobactin fragment 3-11 and/or lysobactin fragment 5-11and/or lysobactin fragment 4-10 and/or lysobactin fragment 1-9.

Preference is given to an enzymatic cleavage of dihydrolysobactin and/oroctahydrolysobactin, whereby a metalloprotease or a cysteine protease isused as enzyme.

Metalloproteases are, for example, thermolysin or mycolysin.

Cysteine proteases are, for example, papain, bromelain or ficin.

The enzymatic cleavage generally takes place in an aqueous cleavagebuffer with addition of a C₁-C₄ alcohol or acetonitrile, preferably in atemperature range from 10° C. to 40° C., preferably in a pH range from 6to 9 under atmospheric pressure.

An aqueous cleavage buffer contains, for example, ammoniumhydrogencarbonate and urea, or sodium phosphate, cysteine and EDTA, orsodium tetraborate, or other additives with which a buffering range ofpH 6 to 9 is covered, preference is given to ammonium hydrogencarbonateand urea.

The C₁-C₄ alcohol is, for example, methanol, ethanol or isopropanol,preference is given to methanol.

Particularly preferably, the enzymatic cleavage takes place in atemperature range from 30° C. to 37° C.

The alcohol concentration in the reaction medium is 0% to 40%,preferably 10% to 15%.

The ratio of enzyme to substrate (dihydrolysobactin and/oroctahydrolysobactin) is 1:1 to 1:4000, preferably 1:25 to 1:100.

The invention further relates to the use of lysobactin fragment 4-11 forthe synthesis of lysobactin derivatives.

These lysobactin derivatives are derivatives in which one or more aminoacids in the ring system of lysobactin are replaced.

The invention further relates to a method for preparing open-chainlysobactin derivatives, in which lysobactin fragment 4-11 is reactedwith a tripeptide having a C-terminal aromatic or hydrophobic amino acidin a buffer medium with addition of a C₁₋₄-alcohol, whereby thetripeptide is present in the form of the free acid or an ester andwhereby the concentration of the C₁₋₄-alcohol in the reaction medium isgreater than 40%.

The C₁-C₄ alcohol is, for example, methanol, ethanol or isopropanol,preference is given to methanol.

Preference is given to a method for the enzymatic synthesis ofopen-chain lysobactin derivatives from lysobactin fragment 4-11 and thetripeptide H-D-X-Y-Phe-OR or H-D-X-Y-(3-cyclohexyl)Ala-OR in a buffermedium with addition of methanol, whereby the methanol concentration inthe reaction medium is greater than 40%,

R represents hydrogen or C₁-C₄-alkyl, preferably ethyl or methyl,particularly preferably methyl,

D-X represents a natural or synthetic (-amino acid in the Dconfiguration and

Y represents a natural or synthetic (-amino acid in the L configuration.

Particular preference is given to a method for the enzymatic synthesisof open-chain lysobactin derivatives from lysobactin fragment 4-11 andthe tripeptide H-D-Leu-Leu-Phe-OMethyl, H-D-Leu-Leu-Phe-OH,H-D-Leu-Leu-(3-cyclohexyl)Ala-OMethyl orH-D-Leu-Leu-(3-cyclohexyl)Ala-OH, whereby chymotrypsin is used as enzymein a buffer medium with addition of methanol, whereby the methanolconcentration in the reaction medium is greater than 40%.

BRIEF DESCRIPTION OF THE DRAWINGS Description of the Figures

FIG. 1: Time course of a preparative enzymatic cleavage withchymotrypsin (Example 11). Superimposition of HPLC diagrams of apreparative enzymatic cleavage with chymotrypsin of a mixture ofdihydro- and octahydrolysobactin. The separation conditions are asreported in the description under Example 30 (UV detection 210 nm).

FIG. 2: Time course of an enzymatic cleavage of octahydrolysobactin withchymotrypsin (Example 5). Superimposition of CZE diagrams of anenzymatic cleavage with chymotrypsin of octahydrolysobactin. Theseparation conditions are as reported in the description under Example31 (UV detection 210 nm).

DEFINITIONS

Dihydrolysobactin:D-Leu-Leu-Phe-Leu(OH)-Leu-D-Arg-Ile-allo-Thr-Gly-Asn(OH)-Ser

Octahydrolysobactin:D-Leu-Leu-Ala(3-cyclohexyl)-Leu(OH)-Leu-D-Arg-Ile-allo-Thr-Gly-Asn(OH)-Ser

Lysobactin fragment 4-11: Leu(OH)-Leu-D-Arg-Ile-allo-Thr-Gly-Asn(OH)-Ser

Lysobactin fragment 1-3: D-Leu-Leu-Phe or D-Leu-Leu-Ala(3-cyclohexyl)

The methods used in the course of the chemical and enzymatic reactionsand analytical characterizations are listed hereinafter.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Examples Abbreviations

aq. aqueous

atm Atmosphere (pressure unit)

conc. concentrated

CZE Capillary zone electrophoresis

DCI Direct chemical ionization (in MS)

DCM Dichloromethane

DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid

EI Electron impact ionization (in MS)

ESI Electrospray ionization (in MS)

h hour(s)

HPLC High pressure, high performance liquid chromatography

HR High resolution

LC-MS Liquid chromatography-coupled mass spectroscopy

LL(3-Cyclohexyl)A D-Leu-Leu-(3-Cyclohexyl)Ala

LLF D-Leu-Leu-Phe

min Minute/minutes

MS Mass spectroscopy

neg. negative

NMR Nuclear magnetic resonance spectroscopy

Pd Palladium

Pd—C Palladium on carbon

pos. positive

PTFE Polytetrafluoroethylene

quant. quantitative

RP-HPLC Reversed-phase HPLC

RT Room temperature

Rt Retention time (in HPLC)

TFA Trifluoroacetic acid

TOF Time of flight

UV Ultraviolet

V is visible

REFERENCES

For the nomenclature of peptides and cyclodepsipeptides c.f.:

-   1. A Guide to IUPAC Nomenclature of Organic Compounds    (Recommendations 1993), 1993, Blackwell Scientific publications.-   2. Nomenclature and symbolism for amino acids and peptides.    Recommendations 1983. IUPAC-IUB Joint Commission on Biochemical    Nomenclature, UK. Biochemical Journal (1984), 219:345-373, as well    as cited literature.

General Methods, LC-MS, HR-MS and HPLC

Method 1 (LC-MS): instrument type MS: Micromass ZQ; instrument typeHPLC: HP 1100 series; UV DAD; column: Phenomenex Synergi 2μ Hydro-RPMercury 20 mm×4 mm; eluent A: 1 l of water+0.5 ml of 50% formic acid,eluent B: 1 l of acetonitrile+0.5 ml of 50% formic acid; gradient: 0.0min 90% A→2.5 min 30% A→3.0 min 5% A→4.5 min 5% A; flow: 0.0 min 1ml/min, 2.5 min/3.0 min/4.5 min 2 ml/min; oven: 50° C.; UV detection:210 nm.

Method 2 (preparative HPLC; symmetry; trifluoroacetic acid): instrument:Gilson Abimed HPLC; UV detector 210 nm; binary pump system; column:SymmetryPrep™C₁₈, Waters, 7 μm; 300×19 mm; eluent A: 0.05%trifluoroacetic acid in water, eluent B: 0.05% trifluoroacetic acid inacetonitrile; gradient: 0-5 min 5% B at flow rate 20 ml/min, 5-30 mingradient ramp from 5 to 60% B with the following increases in flow rate:22 ml/min from 6 min, 23 ml/min from 10 min, 24 ml/min from 15 min;30-35 min gradient ramp from 60% to 98% B with flow rate reduction to 21ml/min from 38 min; 40-45 min 10% B.

Method 3 (Method for preparative separation of dihydro- andoctahydrolysobactin by HPLC): column: SymmetryPrep™ C₁₈, Waters, 7 μm300×19 mm; flow 25 ml/min; RT; eluent A: 0.2% TFA in water, eluent B:acetonitrile, 0-10 min gradient: 80% A, 20% B to 35% A, 65% B; 10.01-15min: 80% A, 20% B; detection 210 nm Fractions monitored by means ofLC-MS (Method 1), freed from acetonitrile on a rotary evaporator andlyophilized.

Method 4 (analytical HPLC 1100, ZQ2, Phenomenex, Synergi, Hydro-RP):instrument type HPLC: HP 1100 Series; UV DAD; column: Phenomenex,MercuryMS, Synergi 2μ Hydro-RP 20×4 mm; eluent A: water/0.05% formicacid, eluent B: acetonitrile; gradient: 0.0-2.5 min, 90-30% A, flow 1-2ml/min, 2.5-3.0 min, 30-5% A, flow 2.0 ml/min, 3.0-4.5 min, 5% A; oven:50° C.; UV detection: 210 nm.

Method 5 (TOF-HR-MS): TOF-HR-MS-ESI+ spectra are recorded using aMicromass LCT instrument (capillary voltage: 3.2 kV, cone voltage: 42 V,source temperature: 120° C., desolvation temperature: 280° C.). For thisa syringe pump (Harvard Apparatus) was used for the sample introduction.Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) is used as standard.

Method 6 (HPLC): instrument type HPLC: HP 1100 Series; UV DAD column:Zorbax Eclipse XBD-C8 (Agilent), 150 mm×4.6 mm, 5 μm; eluent A: 5 ml ofHClO₄/1 of water, eluent B: acetonitrile; gradient: 0-1 min 10% B, 1-4min 10-90% B, 4-5 min 90% B; flow: 2.0 ml/min; oven: 30° C.; UVdetection: 210 and 254 nm.

Method 7 (HPLC): column: Kromasil RP-18, 60 mm×2 mm, 3.5 μm; eluent A: 5ml of HClO₄/1 of water, eluent B: acetonitrile; gradient: 0 min 2% B,0.5 min 2% B, 4.5 min 90% B, 9 min 90% B; flow: 0.75 ml/min; oven: 30°C.; UV detection: 210 nm.

Method 8 (HPLC): column: Kromasil RP-18, 250 mm×4 mm, 5 μm; eluent A: 5ml of HClO₄/1 of water, eluent B: acetonitrile; gradient: 0 min 5% B, 10min 95% B; flow: 1 ml/min; oven: 40° C.; UV detection: 210 nm.

Method 9 (HPLC): column: Kromasil RP-18, 250 mm×4 mm, 5 μm; eluent A: 2ml of HClO₄/1 of water, eluent B: acetonitrile; isocratic: 45% B, 55% A;flow: 1 ml/min; oven: 40° C.; UV detection: 210 nm.

Method 10 (HPLC): instrument: Agilent 1100 with DAD (G1315B), binarypump (G1312A), autosampler (G1313A), solvent degasser (G1379A) andcolumn thermostat (G1316A); column: Agilent Eclipse XDB-C8 4.6×150×5 mm;column temperature: 40° C.; eluent A: 0.05% of 70% perchloric acid inwater; eluent B: methanol; flow: 2.00 ml/min; isocratic: 0-7 min 55% B.

Method 11 (HPLC): analytical HPLC method bromelain/chymotrypsincleavage. About 20 μg of the enzymatic cleavage products or of thestarting compounds are chromatographed on a 300SB-C18 column (4.6 mm×125mm; 3.5 μm material; 300 Angström pore diameter). As eluent, anacetonitrile/TFA gradient is used. Eluent A: 0.1% TFA in water, eluentB: 0.1% TFA in 60% acetonitrile/40% water; gradient: 0 min 0% B, 2 min10% B, 50 min 80% B, 52 min 100% B, 55 min 0% B, 60 min 0% B; flow: 0.7ml/min; column temperature: 40° C.; detection: 210 nm.

Proteinchemical Characterization of Dihydro-, Ocatahydrolysobactin andthe Enzymatic Cleavage Products

Instruments

The sequence analyses are carried out using a protein sequencer Procise™from Applied Biosystems. The standard sequencing program is used. Thesequencer, the various sequencing programs as well as the PTH detectionsystem are described in detail in the operating handbook User's ManualSet, Protein Sequencing System Procise™ (1994), Applied BiosystemsForster City, Calif. 94404, U.S.A.

The reagents for operating the sequencer and the HPLC column for the PTHdetection are obtained from Applied Biosystems.

The HPLC analyses are carried out using an HP1100 HPLC system fromAgilent. A Zorbax 300SB-C18 column (4.6 mm×150 mm; 3.5 μm material; 300Angström pore diameter) from Agilent (D-Waldbronn) is used for theseparations.

The reagents used are of HPLC quality and are obtained from Merck(D-Darmstadt).

The capillary electrophoresis model 270A-HT is from Applied Biosystems.The samples are generally injected hydrodynamically over various timeperiods. The capillary column used (50 μm diameter×72 cm in length) isfrom Applied Biosystems. Separation programs and the function of theanalyzer are described extensively in the handbook of the instrument(User's manual capillary electrophoresis system model 270A HT; AppliedBiosystems Forster City, Calif. 94404, U.S.A.; 1989).

The reagents used are of biochemical quality and are obtained from Merck(D-Darmstadt) or Sigma (D-Deisenhofen).

The amino acid analyses are carried out using an LC3000 amino acidanalyzer from Eppendorf/Biotronik A slightly modified standardseparation program from Eppendorf/Biotronik is used. The separationprograms and the function of the analyzer are extensively described inthe instrument handbook (Handbuch des Aminosäureanalysators LC 3000[handbook of the LC 3000 amino acid analyzer], Wissenschaftliche GeräteGmbH Biotronik, Maintal, 1996).

The reagents used are of biochemical quality and are obtained from Merck(D-Darmstadt), Fluka (D-Neu-Ulm) or Sigma (D-Deisenhofen).

The molecular weights are determined using a ZQ-1 system from Micromass(Manchester, UK). The fragments are thereby separated by means ofRP-18-HPLC chromatography (HP1100 system) and the molecular weight isdetermined by electron spray ionization (ESI). External calibration iscarried out. The calibration and functioning of the systems areextensively described in the handbook of the instrument.

The enzymes and chemicals used are of biochemical quality and areobtained from Fluka, Calbiochem (D-Heidelberg) and Sigma.

The material for the preparative chromatography source 15RPC is obtainedfrom Amersham Bioscience (D-Freiburg). The preparative separation iscarried out using an ÄKTA™ system from Amersham Bioscience.

The chemical compounds mentioned in the invention can also be in theform of salts, solvates or solvates of the salts.

Salts preferred for purpose of the present invention are physiologicallyacceptable salts of the compounds which can be prepared or are useableaccording to the invention. However, also comprised are salts which arenot themselves suitable for pharmaceutical applications, but can beused, for example, for the isolation or purification of the compoundswhich can be prepared or are useable according to the invention, ormixed salts.

Physiologically acceptable salts of the compounds which can be preparedor are useable according to the invention comprise acid addition saltsof mineral acids, carboxylic acids and sulfonic acids, for example saltsof hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid,trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malicacid, citric acid, fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the compounds which can be preparedor are useable according to the invention also comprise salts of usualbases such as, for example, and preferably, alkali metal salts (forexample sodium and potassium salts), alkaline earth metal salts (forexample calcium and magnesium salts) and ammonium salts, derived fromammonia or organic amines having 1 to 16 carbon atoms, such as, forexample, and preferably ethylamine, diethylamine, triethylamine,ethyldiisopropylamine, monoethanolamine, diethanolamine,triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine,dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine andN-methylpiperidine.

Solvates, for the purpose of the invention refer to those forms of thecompounds which can be produced or are useable according to theinvention which, which in solid or liquid state, form a complex bycoordination with solvent molecules. Hydrates are a special form ofsolvates in which the coordination takes place with water.

Example 1D-Leucyl-N1-{(3S,6S,12S,15S,18R,21S,24S,27S,28R)-6-[(1S)-2-amino-1-hydroxy-2-oxoethyl]-18-(3-{[amino(imino)methyl]amino}propyl)-12-[(1S)-1-hydroxyethyl]-3-(hydroxymethyl)-24-[(1R)-1-hydroxy-2-methylpropyl]-21-isobutyl-15-[(1S)-1-methylpropyl]-2,5,8,11,14,17,20,23,26-nonaoxo-28-phenyl-1-oxa-4,7,10,13,16,19,22,25-octaazacyclo-octacosan-27-yl}-L-leucinamidebistrifluoroacetateD-Leucyl-L-leucyl-[(3R)-3-hydroxy-L-phenylalanyl)]-[(3R)-3-hydroxy-L-leucyl]-L-leucyl-D-arginyl-L-isoleucyl-L-allothreonyl-glycyl-[(3S)-3-hydroxy-L-asparaginyl]-L-serine-C1.¹¹-O3.³-lactonebistrifluoroacetate} (lysobactin)

Fermentation:

Culture Medium:

YM: yeast-malt agar: D-glucose (4 g/l), yeast extract (4 g/l), maltextract (10 g/l), 1 liter of Lewatit water. Before sterilization (20minutes at 121° C.), the pH is adjusted to 7.2.

HPM: mannitol (5.4 g/l), yeast extract (5 g/l), meat peptone (3 g/l).

Working preserve: The lyophilized strain (ATCC 53042) is grown in 50 mlof YM medium.

Flask fermentation: 150 ml of YM medium or 100 ml of HPM medium in a 1 lErlenmeyer flask are inoculated with 2 ml of the working preserve andallowed to grow on a shaker at 240 rpm for 30-48 hours at 28° C.

30 l fermentation: 300 ml of the flask fermentation (HPM medium) areused to inoculate a sterile 30 l nutrient medium solution (1 ml ofantifoam SAG 5693/1). This culture is allowed to grow for 21 hours at28° C., 300 rpm and aeration with sterile air of 0.3 vvm. The pH is keptconstant at Ph=7.2 with 1 M hydrochloric acid. In total, 880 ml of 1 Mhydrochloric acid are added during the culturing period.

Main culture (200 l): 15×150 ml of YM medium in 1 l Erlenmeyer flasksare inoculated with 2 ml of the working preserve and allowed to grow onthe shaker at 28° C. for 48 hours and at 240 rpm. 2250 ml of thisculture are used to inoculate a sterile 200 l nutrient media solution(YM) (1 ml of antifoam SAG 5693/1) and it is allowed to grow for 18.5hours at 28° C., 150 rpm and aeration with sterile air of 0.3 vvm.

Hourly samples (50 ml) are taken to check the course of thefermentation. 1 ml of methanol (0.5% trifluoroacetic acid) is added to 2ml of this culture broth and the mixture is filtered through a 0.45 μmfilter. 30 μl of this suspension are analyzed means of by HPLC (Method 6and Method 7).

After 18.5 hours, the culture broth of the main culture is separatedinto supernatant and sediment at 17 000 rpm.

Isolation:

The supernatant (183 l) is adjusted to pH 6.5(7 using concentratedtrifluoroacetic acid or a sodium hydroxide solution and loaded onto aLewapol column (OC 1064, 60 l contents). Elution is subsequently carriedout with pure water, water/methanol 1:1 and subsequently with puremethanol (containing 0.1% trifluoroacetic acid). This organic phase isconcentrated in vacuo to a residual aqueous residue of 11.5 l.

The residual aqueous phase is bound to silica gel C₁₈ and separated(MPLC, Biotage Flash 75, 75×30 cm, KP-C18-WP, 15-20 μm, flow: 30 ml;eluent: acetonitrile/water containing 0.1% trifluoroacetic acid;gradient: 10%, 15% and 40% acetonitrile). The 40% acetonitrile phasewhich contains the main amount of Example 1A, is concentrated in vacuoand subsequently lyophilized (about 13 g). This mixture of solids isseparated in 1.2 g portions, first on a preparative HPLC (Method 1),subsequently by gel filtration on Sephadex LH-20 (5×70 cm,acetonitrile/water 1:1, in each case containing 0.05% trifluoroaceticacid) and a further preparative HPLC (Method 8).

This process yields 2250 mg of Example 1.

The sediment is taken up in 41 of acetone/water 4:1, 2 kg of Celite areadded, the mixture is adjusted to pH=6 using trifluoroacetic acid,stirred and centrifuged. The solvent is concentrated in vacuo and theresidue is freeze dried. The lyophilizate obtained (89.9 g) is taken upin methanol, filtered, concentrated and separated on silica gel (Method9). Example 1A is then purified by gel filtration (Sephadex LH-20, 5×68cm, water/acetonitrile 9:1 (containing 0.05% trifluoroacetic acid),flow: 2.7 ml/min, fraction size 13.5 ml) to give the pure substance.

This process yields 447 mg of Example 1.

HPLC (Method 6): R_(t)=6.19 min

MS (ESIpos): m/z=1277 [M+H]⁺

1H NMR (500.13 MHz, d₆-DMSO): δ=0.75 (d, 3H), 0.78 (d, 6H), 0.80 (t,3H), 0.82 (d, 3H), 0.90 (d, 3H), 0.91 (d, 3H), 0.92 (d, 3H), 0.95 (d,3H), 0.96 (d, 3H), 1.05 (m, 1H), 1.19 (d, 3H), 1.25 (m, 2H), 1.50 (m,4H), 1.51 (m, 2H), 1.55 (m, 1H), 1.61 (m, 1H), 1.65 (m, 1H), 1.84 (m,1H), 1.85 (m, 1H), 1.86 (m, 1H), 1.89 (m, 1H), 1.95 (m, 1H), 2.75 (m,2H), 3.40 (m, 1H), 3.52 (m, 2H), 3.53 (dd, 1H), 3.64 (m, 2H), 3.66 (m,1H), 3.68 (dd, 1H), 3.73 (m, 2H), 4.00 (dd, 1H), 4.02 (br., 1H), 4.13(br., 1H), 4.32 (dd, 1H), 4.39 (t, 1H), 4.55 (m, 1H), 4.75 (dd, 1H),5.19 (t, 1H), 5.29 (d, 1H), 5.30 (br., 1H), 5.58 (m, 2H), 6.68 (m, 3H),6.89 (d, 1H), 6.93 (m, 3H), 6.94 (br., 1H), 6.98 (d, 1H), 7.12 (br.,1H), 7.20 (br., 2H), 7.23 (m, 2H), 7.42 (m, 2H), 7.54 (d, 1H), 7.58 (d,1H), 8.32 (br., 1H), 9.18 (br., 1H), 9.20 (m, 2H), 9.50 (br., 1H).

¹³C-NMR (125.77 MHz, d₆-DMSO): δ=10.3, 15.3, 19.0, 19.2, 19.6, 20.0,20.9, 22.0, 22.4, 23.0, 23.2, 24.3, 24.4, 25.0, 25.4, 26.0, 27.8, 30.9,35.4, 39.5, 40.8, 40.9, 41.6, 44.1, 51.5, 52.7, 55.9, 56.2, 56.4, 57.9,58.8, 60.2, 61.1, 62.6, 70.1, 71.6, 71.7, 75.5, 128.1, 128.6, 136.7,156.8, 168.2, 170.1, 170.4, 171.2, 171.5, 171.9, 172.2, 172.4, 173.7.

The assignment of the signals was carried out according to theassignment described in the literature (T. Kato, H. Hinoo, Y. Terui, J.Antibiot. (1988) 61:719-725).

Example 2 and Example 3D-Leu-Leu-Phe-[(3R)-Leu(3-OH)]-Leu-D-Arg-Ile-aThr-Gly-[(3S)-3-Asn(3-OH)]-Ser-trifluoroacetate(dihydrolysobactin) andD-Leu-Leu-Ala(3-cyclohexyl)-[(3R)-Leu(3-OH)]-Leu-D-Arg-Ile-aThr-Gly-[(3S)-3-Asn(3-OH)]-Ser-trifluoroacetate(octahydrolysobactin)

Hydrogenation Method 1:

The compound from Example 1 (lysobactin, 250 mg, 170 mmol) is dissolvedin isopropanol/water (2:1, 60 ml) and hydrogenated under 1 atm ofhydrogen in the presence of 200 mg of Pd (10% on carbon). The course ofthe reaction is followed by means of LC-MS (Method 1). After virtuallycomplete conversion (>95%), the catalyst is filtered off, washed withisopropanol and the filtrate is lyophilized. In this crude product,according to LC-MS, the products are distributed as follows:dihydrolysobactin about 74%, octahydro-lysobactin about 12%. The residueis purified by HPLC (Method 2). After lyophilization of the suitablefractions, the pure compound Example 2 is obtained (81.5 mg, 31% oftheory).

LC-MS: (Method 1): R_(t)=1.56 min ES⁺: m/z=1279 [M+H]⁺, 640.1 [M+2H]²⁺;ES⁻: m/z=1277 [M-H]⁻, 638.1 [M-2H]²⁻.

See Table 1 for the peptide sequences of the hydrolysobactins.

Hydrogenation Method 2:

By hydrogenation under a hydrogen pressure of 3 atm, in a methodotherwise identical to hydrogenation method 1, the followingdistribution in the crude product determined by LC-MS is obtained:dihydrolysobactin about 80%, octahydrolysobactin about 17%. After HPLCpurification (Method 2), the pure compound Example 2 is obtained (86 mg,33% of theory).

Hydrogenation Method 3:

With a prolonged hydrogenation period at 3 bar hydrogen or using ahigher pressure (up to 80 bar hydrogen pressure), proportionately moreoctahydrolysobactin can be obtained. In most cases, the crude mixturesof dihydro- and octahydrolysobactin are not separated, but are useddirectly in the enzymatic cleavage.

Hydrogenation Method 4:

In the following case the compound octahydrolysobactin is also isolatedin pure form:

Lysobactin (Example 1, 1.04 g, 0.69 mmol) is dissolved inisopropanol/water (2:1, 90 ml) and hydrogenated under 3 atm hydrogen for7 days in the presence of 200 mg of Pd (10% on carbon). The catalyst isfiltered off, washed with isopropanol and the filtrate is freed fromisopropanol on a rotary evaporator and then lyophilized. In this crudeproduct the products are distributed according to LC-MS (Method 1) asfollows: dihydrolysobactin about 65%, octa-hydrolysobactin about 35%.The residue is purified by HPLC (Method 2, subsequently Method 3).Dihydrolysobactin (Example 2) (280 mg, 27% of theory) andoctahydrolysobactin (Example 3) (212 mg, 20% of theory) are obtained.

LC-MS: (Method 1): R_(t)=1.63 min ESIpos.: m/z=643.3 (100) [M+2H]²⁺:ESIneg.: m/z=1283 [M-H]⁺, 641.2 [M-2H]²⁻.

Hydrogenation Method 5:

As an example of a hydrogenation under high pressure hydrogen, after 4days at 40° C. and 50 bar hydrogen, the following crude mixture isobtained according to LC-MS (Method 1): 45% dihydrolysobactin and 45%octahydrolysobactin.

Hydrogenation Method 6:

Lysobactin bistrifluoroacetate (Example 1, 500 mg, 0.33 mmol) isdissolved in isopropanol/water 2:1 (30 ml). Under an argon protectivegas atmosphere, 10 percent palladium on carbon (100 mg) is added. Thereaction mixture is stirred (after degassing) in a pressure autoclave at80-70 bar hydrogen and RT for 48 h. 10% palladium on carbon (100 mg) isagain added to the reaction. The reaction mixture is (after degassing)again stirred in a pressure autoclave at 80-70 bar hydrogen and RT for48 h. Now no lysobactin is detectable any more by means of HPLC (forexample Method 4). The reaction mixture is filtered through a glass frit(pore size 2 or 3), concentrated in vacuo, again taken up inmethanol/0.2% glacial acetic acid, filtered through a syringe filter(Biotage, PTFE), concentrated in vacuo and dried under high vacuum. 496mg (quant.) of product (80% dihydrolysobactin, 20% octahydrolysobactin)are obtained.

Hydrogenation Method 7:

Lysobactin monotrifluoroacetate monoacetate (5 mg, 3.45 mmol) ishydrogenated in a mixture of isopropanol (2 ml), water (0.25 ml) andacetic acid (0.05 ml) in the presence of platinum dioxide (20 mg) at 80bar and 50° C. After 17 h, the pressure is relieved, the system isvented with argon and the suspension freed from the catalyst by means ofa microfilter. LC-MS analysis of the filtrate (Method 4) shows 7% oftheory octahydrolysobactin (R_(t)=1.54 min, Method 4).

Hydrogenation Method 8:

Lysobactin bistrifluoroacetate (Example 1A, 10 g, 6.65 mmol) isdissolved in isopropanol/water 9:2 (110 ml). Under an argon protectivegas atmosphere, palladium on carbon (10%; 5 g) is added. The reactionmixture (after degassing) is stirred in a pressure autoclave at 80-70bar hydrogen pressure and 40° C. for 12 h. Palladium on carbon (10%; 5g) is again added to the reaction. The reaction mixture (afterdegassing) is again stirred in a pressure autoclave at 80-70 barhydrogen pressure and 40° C. for 12 h. The reaction mixture (afterdegassing) is once again stirred in a pressure autoclave at 80-70 barhydrogen pressure and 40° C. for 12 h. Now no lysobactin is detectableany more by means of analytical HPLC (Method 10). The reaction mixtureis filtered through kieselguhr, concentrated in vacuo and dried under ahigh vacuum. 9.17 g (99% of theory) of product (60% dihydrolysobactin,40% octahydrolysobactin) are obtained.

Hydrogenation Method 9:

Lysobactin bistrifluoroacetate (Example 1A, 5 g, 3.32 mmol) is dissolvedin isopropanol/water 9:2 (110 ml). Under an argon protective gasatmosphere, palladium on carbon (10%; 5 g) is added. The reactionmixture (after degassing) is stirred in a pressure autoclave at 80 barhydrogen pressure and 40° C. for 12 h. The reaction mixture is filteredthrough kieselguhr, concentrated in vacuo and dried under high vacuum.The hydrogenation is repeated a further three times each time using 5.0g of lysobactin bistrifluoroacetate (in total: 4 passes). As combinedproduct fraction 18.27 g of product(dihydro-lysobactin:octahydrolysobactin, about 5:4) are obtained.

Example 4 Chymotrypsin Cleavage of Dihydrolysobactin, Enzyme/SubstrateRatio 1:50

200 μg of dihydrolysobactin are dissolved in 10 μl of methanol and then190 μl of cleavage buffer (0.1 M ammonium hydrogencarbonate/0.5 M ureapH 8) are added. 4 μg of: chymotrypsin (1:50) are added and the reactionis carried out at 37° C. Aliquots of 30 μl are taken after 0, 0.5, 1, 3,6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis byHPLC, capillary zone electrophoresis, sequence analysis, amino acidanalysis, or MS study.

See Table 2 the for the peptide sequences of the chymotrypsin cleavageproducts.

Example 5 Chymotrypsin Cleavage of Octahydrolysobactin, Enzyme SubstrateRatio 1:50

200 μg of octahydrolysobactin are dissolved in 10 μl of methanol andthen 190 μl of cleavage buffer (0.1 M ammonium hydrogencarbonate/0.5 Murea pH 8) are added. 4 μg of: chymotrypsin (1:50) are added and thereaction is carried out at 37° C. Aliquots of 30 μl are taken after 0,0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 2 for the peptide sequences of the chymotrypsin cleavageproducts.

Example 6 Analytical Chymotrypsin Cleavage of the MixtureDihydro-/Octahydrolysobactin, Enzyme Substrate Ratio 1:25

200 μg of dihydro- (59%) and octahydrolysobactin (34%) are dissolved in10 μl of methanol and then 190 μl of cleavage buffer (0.1 M ammoniumhydrogencarbonate/0.5 M urea pH 8) are added. 8 μg of chymotrypsin(1:25) are added and the reaction is carried out at 37° C. Aliquots of30 μl are taken after 0, 0.5, 1, 3 h and the enzyme cleavage is stoppedwith 30 μl of acetonitrile/1% TFA. The samples are stored at ˜20° C.until analysis.

See Table 2 for the peptide sequences of the chymotrypsin cleavageproducts.

Example 7 Analytical Chymotrypsin Cleavage of the MixtureDihydro-/Octahydrolysobactin, Enzyme Substrate Ratio 1:400

150 μg of dihydro-(59%) and octahydrolysobactin (34%) are dissolved in15 μl of ethanol and then 126 μl of cleavage buffer (0.1 M ammoniumhydrogencarbonate/0.5 M urea pH 8) are added. 0.38 μg of chymotrypsin (9μl of chymotrypsin solution water/ethylene glycol/cleavage buffer, 0.2mg/ml; 1:400) are added and the reaction is carried out at 37° C.Aliquots of 25 μl are taken after 0, 0.5, 1, 3 h and the enzyme cleavageis stopped with 25 μl of 30% acetonitrile/0.1% TFA. The samples arestored at −20° C. until analysis.

See Table 2 for the peptide sequences of the chymotrypsin cleavageproducts.

Example 8 Analytical Chymotrypsin Cleavage of the MixtureDihydro-/Octahydrolysobactin Substrate Concentration 6 mg/ml

900 μg of dihydro- (59%) and octahydrolysobactin (34%) are dissolved in15 μl of methanol and then 99 μl of cleavage buffer (0.1 M ammoniumhydrogencarbonate/0.5 M urea pH 8) are added. 36 μg of chymotrypsin (36μl of chymotrypsin solution water/ethylene glycol 1:1, 1 mg/ml; 1:25)are added and the reaction is carried out at 37° C. Aliquots of 25 μlare taken after 0, 0.5, 1, 3 h and the enzyme cleavage is stopped with25 μl of 30% acetonitrile/0.1% TFA. The samples are stored at −20° C.until analysis.

See Table 2 for the peptide sequences of the chymotrypsin cleavageproducts.

Example 9 Analytical Chymotrypsin Cleavage of the MixtureDihydro-/Octahydrolysobactin Solvent Concentration 30% Methanol

150 μg of dihydro- (59%) and octahydrolysobactin (34%) are dissolved in45 μl of methanol and then 99 μl of cleavage buffer (0.1 M ammoniumhydrogencarbonate/0.5 M urea pH 8) are added. 6 μg of chymotrypsin (6 μlof chymotrypsin solution water/ethylene glycol 1:1, 1 mg/ml; 1:25) areadded and the reaction is carried out at 37° C. Aliquots of 25 μl aretaken after 0, 0.5, 1, 3 h and the enzyme cleavage is stopped with 25 μlof 30% acetonitrile/0.1% TFA. The samples are stored at −20° C. untilanalysis.

See Table 2 for the peptide sequences of the chymotrypsin cleavageproducts.

Example 10 Analytical Chymotrypsin Cleavage of the MixtureDihydro-/Octahydrolysobactin Cleavage at Room Temperature

200 μg of dihydro- (59%) and octahydrolysobactin (34%) are dissolved in10 μl of methanol and then 190 μl of cleavage buffer (0.1 M ammoniumhydrogencarbonate/0.5 M urea pH 8) are added. 8 μg of chymotrypsin (8 μlof chymotrypsin solution water/ethylene glycol 1:1, 1 mg/ml; 1:25) areadded and the reaction is carried out at room temperature (20-25° C.).Aliquots of 30 μl are taken after 0, 0.5, 1, 3, 6 h and the enzymecleavage is stopped with 30 μl of 30% acetonitrile/1% TFA. The samplesare stored at −20° C. until analysis.

See Table 2 for the peptide sequences of the chymotrypsin cleavageproducts.

Example 11 Fragment 4-11[(3R)-Leu(3-OH)]-Leu-D-Arg-Ile-aThr-Gly-[(3S)-3-Asn(3-OH)]-SerTrifluoroacetate

Preparative Chymotrypsin Cleavage of Dihydrolysobactin SubstrateConcentration 1 mg/ml

2×80 mg of dihydrolysobactin (35.3 mmol and 33.8 mmol of pure peptidedetermined by amino acid analysis) are dissolved in 8 ml of methanoleach and then 69 ml of cleavage buffer (0.1 M ammoniumhydrogencarbonate/0.5 M urea pH 8) each are added. Before the additionof enzyme, the solutions are warmed to 37° C. in a drying cabinet. 3.2mg of chymotrypsin (3.2 ml of chymotrypsin solution water/ethyleneglycol 1:1, 1 mg/ml; 1:25; preheated to 37° C.) are added and thereactions are carried out at 37° C. Aliquots of 200 μl are taken after0.5, 1 h and the enzyme cleavages are stopped with 200 μl of 30%acetonitrile/0.1% TFA. The samples are analyzed by HPLC in parallel tothe enzyme cleavages within 15 min (retention time fragment 4-11 about3.6 min, fragment 1-3 (LLF) about 9.6 min, conditions: solvent A 0.1%TFA, solvent B 60% acetonitrile/0.1% TFA, gradient: 0 min 30% B, 10 min80% B, 11 min 100% B, 12 min 30% B, 15 min 30% B; flow 0.7 ml/min, 40°C., UV detection 210 nm). The enzyme reactions are stopped after about70 min with 3 ml of acetonitrile and about 0.6 ml of TFA. The pH of thesolution is between 1 and 2. The solutions can be stored at −20° C.until the preparative separation.

Preparative Separation of Fragments 1-3 and 4-11

2× about 80 ml of the cleavage solutions are filtered through a filter(0.2 μm) and then combined. The solution is divided into four portionseach of about 38.5 ml (total 154 ml) and each is chromatographed on aSource 15RPC column (3 ml) using an acetonitrile/TFA gradient.Conditions: solvent A 0.1% TFA, solvent B 0.1% TFA/acetonitrile;gradient: 0% B to 45% B in 40 min; flow 2 ml/min; UV detection 210 nm.The four runs are carried out sequentially and the fractions arecollected in the same tube. The resultant chromatograms are identical.

Fragments 4-11 (Rt=about 15 min) and 1-3 (LLF) (Rt=about 25 min) arecombined, diluted 1:1 with water and then lyophilized.

200 μl aliquots of the respective pools are lyophilized separately foramino acid analysis, analytical HPLC, capillary zone electrophoresis(CZE), sequence analysis and mass spectrometry.

The yield of fragment 4-11 according to amino acid analysis is 68.3 mmol(99% of theory) and of fragment 1-3 67.4 mmol (98% of theory).

Example 12 Preparative Chymotrypsin Cleavage of the MixtureDihydro/Octahydrolysobactin 1 mg/ml Batch 1

2×700 mg of dihydro- (56%) and octahydrolysobactin (21%) (682 mmol ofdihydro- and octahydrolysobactin present as pure peptides determined byamino acid analysis) are dissolved in 70 ml of methanol each and then602 ml of cleavage buffer (0.1 M ammonium hydrogencarbonate/0.5 M ureapH 8) each are added. Before the addition of enzyme the solutions arewarmed to 37° C. in a drying cabinet. 28 mg of chymotrypsin (28 ml ofchymotrypsin solution water/ethylene glycol 1:1, 1 mg/ml; 1:25; 37° C.preheated) are added and the reactions are carried out at 37° C.Aliquots of 200 μl are taken after 0.5, 1 h and the enzyme cleavages arestopped with 200 μl of 30% acetonitrile/0.1% TFA. The samples areanalyzed by HPLC in parallel to the enzyme cleavages within 15 min(retention time fragment 4-11 about 3.6 min, fragment 1-3 (LLF) about9.6 min, fragment 1-3 (LL(3-cyclohexyl)A) about 11.3 min, conditions:solvent A 0.1% TFA, solvent B 60% acetonitrile/0.1% TFA, gradient: 0 min30% B, 10 min 80% B, 11 min 100% B, 12 min 30% B, 15 min 30% B; flow 0.7ml/min, 40° C., UV detection 210 nm). The enzyme reactions are stoppedafter about 60 min with 30 ml of acetonitrile and about 6 ml of TFA. ThepH of the solution is between 1 and 2. The solutions can be stored at−20° C. until the preparative separation.

Batch 2

775 mg of dihydro- (45%) and octahydrolysobactin (48%) (468 mmol ofdihydro- and octahydrolysobactin present as pure peptides determined byamino acid analysis) are dissolved in 77.5 ml of methanol and then 667ml of cleavage buffer (0.1M ammonium hydrogencarbonate/0.5M urea pH 8)are added. Before the addition of the enzyme the solution is warmed to37° C. in a drying cabinet. 31 mg of chymotrypsin (31 ml of chymotrypsinsolution water/ethylene glycol 1:1, 1 mg/ml; 1:25; 37° C. preheated) areadded and the reaction is carried out at 37° C. Aliquots of 200 μl aretaken after 0.5, 1 h and the enzyme cleavage is stopped with 200 μl of30% acetonitrile/0.1% TFA. The samples are analyzed by HPLC in parallelto the enzyme cleavage within 15 min (retention time fragment 4-11 about3.6 min, fragment 1-3 (LLF) about 9.6 min, fragment 1-3(LL(3-cyclohexyl)A) about 11.3 min) (solvent A 0.1% TFA, solvent B 60%acetonitrile/0.1% TFA, gradient: 0 min 30% B, 10 min 80% B, 11 min 100%B, 12 min 30% B, 15 min 30% B; flow 0.7 ml/min, temperature: 40° C., UVdetection 210 nm). The enzyme reaction is stopped after 60 min with 30ml of acetonitrile and about 6 ml of TFA. The pH of the solution shouldbe between 1 and 2. The solution can be stored at −20° C. until thepreparative separation.

Preparative Separation of Fragments 1-3 and 4-11

The cleavage batches 1 and 2 are filtered through a filter (0.2 μm) andthen combined. The solution is divided into several portions and each ischromatographed on a Source 15RPC column using an acetonitrile/TFAgradient as described above. The runs are carried out successively andthe fractions collected in the same tube. The resultant chromatogramsare identical.

Fragment 4-11 (Rt. about 15 min) is combined, diluted 1:1 with water andthen lyophilized.

The yield of fragment 4-11, after lyophilization, is 1.1 g (1095 mmol).For a starting amount of 1150 mmol of cleavable material, the yield offragment 4-11 is 95% of theory.

Example 13 Preparative Chymotrypsin Cleavage of the MixtureDihydro/Octahydrolysobactin Substrate Concentration 3 mg/ml

2×0.995 g of a mixture of dihydro- (52%) and octahydrolysobactin (37%)are dissolved in 33 ml of methanol each and then 257 ml of cleavagebuffer (0.1M ammonium hydrogencarbonate/0.5 M urea pH 8) each are added.Before the addition of the enzyme the solution is warmed to 37° C. in adrying cabinet. 39.6 mg of chymotrypsin (39.6 ml of chymotrypsinsolution water/ethylene glycol 1:1, 1 mg/ml; 1:25; 37° C. preheated) areadded and the reaction is carried out at 37° C. Aliquots of 200 μl aretaken after 0.5, 1 h and the enzyme cleavage is stopped with 200 μl of30% acetonitrile/0.1% TFA. The samples are analyzed by HPLC in parallelto the enzyme cleavage within 15 min (retention time fragment 4-11 about3.6 min, fragment 1-3 (LLF) about 9.6 min, fragment 1-3(LL(3-cyclohexyl)A) about 11.3 min) (solvent A 0.1% TFA, solvent B 60%acetonitrile/0.1% TFA, gradient: 0 min 30% B, 10 min 80% B, 11 min 100%B, 12 min 30% B, 15 min 30% B; flow: 0.7 ml/min, temperature: 40° C., UVdetection 210 nm). The enzyme reactions are stopped after 60 min with 30ml of acetonitrile and about 2.5 ml of TFA each. The pH of the solutionshould be between 1 and 2. The solution can be stored at −20° C. untilthe preparative separation.

Example 14 Preparative Chymotrypsin Cleavage of the MixtureDihydro/Octahydrolysobactin Substrate Concentration 5 mg/ml

10 g of dihydro- (about 40%) and octahydrolysobactin (about 60%) aredissolved in 200 ml of methanol and then 1700 ml of cleavage buffer (0.1M ammonium hydrogen-carbonate/0.5 M urea pH 8) are added. Before theaddition of the enzyme the solution is warmed to 37° C. in a dryingcabinet. 400 mg of chymotrypsin (100 ml of chymotrypsin solutionwater/ethylene glycol 1:1, 4 mg/ml; 1:25; 37° C. preheated) are addedand the reaction is carried out at 37° C. Aliquots of 200 μl are takenafter 0.5, 1 h and the enzyme cleavage is stopped with 200 μl of 30%acetonitrile/0.1% TFA. The samples are analyzed by HPLC in parallel tothe enzyme cleavage within 15 min (retention time fragment 4-11 about3.6 min, fragment 1-3 (LLF) about 9.6 min, fragment 1-3(LLA(3-cyclohexyl)) about 11.3 min) (solvent A 0.1% TFA, solvent B 60%acetonitrile/0.1% TFA, gradient 0 min 30% B, 10 min 80% B, 11 min 100%B, 12 min 30% B, 15 min 30% B; flow 0.7 ml/min, temperature: 40° C., UVdetection 210 nm). The enzyme reaction is stopped after 60 min with 75ml of acetonitrile and about 15 ml of TFA. The pH of the solution shouldbe between 1 and 2. The solution can be stored at −20° C. until thepreparative separation.

Fragment 4-11 is isolated as described above by preparative HPLC inseveral runs.

The activity of the chymotrypsin batch used (70 U/mg) is checked by acontrol cleavage using the protein interleukin-4 double muteinArg(121)→Asp(121)/Tyr(124)→Asp(124) (BAYER Healthcare AG, D-Wuppertal).

Example 15 Subtilisin Cleavage of Dihydrolysobactin

200 μg of dihydrolysobactin are dissolved in 10 μl of methanol and then190 μl of cleavage buffer (0.1 M ammonium hydrogencarbonate/0.5 M ureapH 8) are added. 4 μg of subtilisin (1:50) are added and the reaction iscarried out at 37° C. Aliquots of 30 μl are taken after 0, 0.5, 1, 3, 6and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 3 for the peptide sequences of the subtilisin cleavageproducts.

Example 16 Subtilisin Cleavage of Octahydrolysobactin

200 μg of octahydrolysobactin are dissolved in 10 μl of methanol andthen 190 μl of cleavage buffer (0.1 M ammonium hydrogencarbonate/0.5 Murea pH 8) are added. 4 μg of subtilisin (1:50) are added and thereaction is carried out at 37° C. Aliquots of 30 μl are taken after 0,0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 3 for the peptide sequences of the subtilisin cleavageproducts.

The activity of the subtilisin batch used (about 12 U/mg) is checked bya control cleavage using the protein interleukin-4 double muteinArg(121)→Asp(121)/Tyr(124)→Asp(124) (BAYER Healthcare AG, D-Wuppertal).

Example 17 Thermolysin Cleavage of Dihydrolysobactin

200 μg of dihydrolysobactin are dissolved in 10 μl of methanol and then190 μl of cleavage buffer (0.1 M tris(hydroxymethyl)aminomethane/5 mMcalcium chloride pH 7.45) are added. 4 μg of thermolysin (1:50) areadded and the reaction is carried out at 37° C. Aliquots of 30 μl aretaken after 0, 0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stoppedwith 30 μl of acetonitrile/1% TFA. The samples are stored at −20° C.until analysis.

See Table 4 for the peptide sequences of the thermolysin cleavageproducts.

Example 18 Thermolysin Cleavage of Octahydrolysobactin

200 μg of octahydrolysobactin are dissolved in 10 μl of methanol andthen 190 μl of cleavage buffer (0.1 M ammonium hydrogencarbonate/0.5 Murea pH 8) are added. 4 μg of thermolysin (1:50) are added and thereaction is carried out at 37° C. Aliquots of 30 μl are taken after 0,0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 4 for the peptide sequences of the thermolysin cleavageproducts.

The activity of the thermolysin batch used (about 55 U/mg) is checked bya control cleavage using the protein interleukin-4 double muteinArg(121)→Asp(121)/Tyr(124)→Asp(124) (BAYER Healthcare AG, D-Wuppertal).

Example 19 Papain Cleavage of Dihydrolysobactin

200 μg of dihydrolysobactin are dissolved in 10 μl of methanol and then190 μl of cleavage buffer (0.1 M sodium phosphate/10 mM cysteine, 2 mMEDTA pH 6.5) are added. 4 μg of papain (1:50) are added and the reactionis carried out at 37° C. Aliquots of 30 μl are taken after 0, 0.5, 1, 3,6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 5 for the peptide sequences of the papain cleavage products.

Example 20 Papain Cleavage of Octahydrolysobactin

200 μg of octahydrolysobactin are dissolved in 10 μl of methanol andthen 190 μl of cleavage buffer (0.1 M sodium phosphate/10 mM cysteine, 2mM EDTA pH 6.5) are added. 4 μg of papain (1:50) are added and thereaction is carried out at 37° C. Aliquots of 30 μl are taken after 0,0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 5 for the peptide sequences of the papain cleavage products.

The activity of the papain batch used (about 11 U/mg) is checked by acontrol cleavage using the protein interleukin-4 double muteinArg(121)→Asp(121)/Tyr(124)→Asp(124) (BAYER Healthcare AG, D-Wuppertal).

Example 21 Proteinase K Cleavage of Dihydrolysobactin

200 μg of dihydrolysobactin are dissolved in 10 μl of methanol and then190 μl of cleavage buffer (0.1 M sodium tetraborate pH 9) are added. 4μg of proteinase K (1:50) are added and the reaction is carried out at37° C. Aliquots of 30 μl are taken after 0, 0.5, 1, 3, 6 and 24 h andthe enzyme cleavage is stopped with 30 μl of acetonitrile/1% TFA. Thesamples are stored at −20° C. until analysis.

See Table 6 for the peptide sequences of the proteinase K cleavageproducts.

Example 22 Proteinase K Cleavage of Octahydrolysobactin

200 μg of octahydrolysobactin are dissolved in 10 μl of methanol andthen 190 μl of cleavage buffer (0.1 M sodium tetraborate pH 9) areadded. 4 μg of proteinase K (1:50) are added and the reaction is carriedout at 37° C. Aliquots of 30 μl are taken after 0, 0.5, 1, 3, 6 and 24 hand the enzyme cleavage is stopped with 30 μl of acetonitrile/1% TFA.The samples are stored at −20° C. until analysis.

See Table 6 for the peptide sequences of the proteinase K cleavageproducts.

The activity of the proteinase K batch used (about 30 U/mg) is checkedby a control cleavage using the protein interleukin-4 double muteinArg(121)→Asp(121)/Tyr(124)→Asp(124) (BAYER Healthcare AG, D-Wuppertal).

Example 23 Bromelain Cleavage of Dihydrolysobactin

200 μg of dihydrolysobactin are dissolved in 10 μl of methanol and then190 μl of cleavage buffer (0.1 M sodium phosphate, 10 mM cysteine, 2 mMEDTA pH 6.5) are added. 4 g of bromelain (1:50) are added and thereaction is carried out at 37° C. Aliquots of 30 μl are taken after 0,0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 7 for the peptide sequences of the bromelain cleavageproducts.

Example 24 Bromelain Cleavage of Octahydrolysobactin

200 μg of octahydrolysobactin are dissolved in 10 μl of methanol andthen 190 μl of cleavage buffer (0.1 M sodium phosphate, 10 mM cysteine,2 mM EDTA pH 6.5) are added. 4 μg of bromelain (1:50) are added and thereaction is carried out at 37° C. Aliquots of 30 μl are taken after 0,0.5, 1, 3, 6 and 24 h and the enzyme cleavage is stopped with 30 μl ofacetonitrile/1% TFA. The samples are stored at −20° C. until analysis.

See Table 7 for the peptide sequences of the bromelain cleavageproducts.

The activity of the bromelain batch used (about 4 U/mg) is checked by acontrol cleavage using the protein interleukin-4 double muteinArg(121)→Asp(121)/Tyr(124)→Asp(124) (BAYER Healthcare AG, D-Wuppertal).

Example 25 Enzymatic Synthesis of Dihydrolysobactin with Chymotrypsin

800 μg of the peptide Leu-Leu-PheOMe and 100 μg of the peptide 4-11 aredissolved in 200 μl of methanol and then 200 μl of synthesis buffer (0.1M sodium tetraborate pH 9) are added. 24 μg of chymotrypsin are addedand the reaction is carried out at 37° C. Aliquots of 30 μl are takenafter 0, 0.5, 1, 3, 6 and 24 h and the synthesis is stopped with 30 μlof acetonitrile/1% TFA. The samples are stored at −20° C. untilanalysis.

Dihydrolysobactin is detected using HPLC and CZE.

Example 26 Enzymatic Synthesis of Dihydrolysobactin Derivatives withChymotrypsin

800 μg of the peptide Boc-Leu-Leu-PheOMe are dissolved in 200 μl oftetrachloromethane and then 200 μl of synthesis buffer (0.1 M sodiumtetraborate pH 9) which contains 100 μg of the peptide 4-11 are added.24 μg of chymotrypsin are added and the reaction is carried out at 37°C. Aliquots of 30 μl are taken after 0, 0.5, 1, 3, 6 and 24 h and thesynthesis is stopped with 30 μl of acetonitrile/1% TFA. The samples arestored at −20° C. until analysis.

Dihydrolysobactin derivatives are detected by HPLC and CZE.

Example 27 Enzymatic Synthesis of Octahydrolysobactin with Chymotrypsin

800 μg of the peptide Leu-Leu-Ala(3-cyclohexyl)OMe and 100 μg of thepeptide 4-11 are dissolved in 200 μl of methanol and then 200 μl ofsynthesis buffer (0.1 M sodium tetraborate pH 9) are added. 24 μg ofchymotrypsin are added and the reaction is carried out at 37° C.Aliquots of 30 μl are taken after 0, 0.5, 1, 3, 6 and 24 h and thesynthesis is stopped with 30 μl of acetonitrile/1% TFA. The samples arestored at −20° C. until analysis.

Octahydrolysobactin is detected by HPLC and CZE.

Example 28 N-Terminal Sequence Analysis

3 nmol of fragments dissolved in 60% acetonitrile/0.1% TFA are loadedonto a sequencer sheet which is preincubated with PolybrenR. Theproteins are sequenced using the usual sequencer cycle. The PTH-aminoacids are identified by means of online HPLC using a 40 pmol PTHstandard. The non-proteinogenic amino acids are identified by theirrelative position to the standard amino acids. The purity of thepeptides is estimated from the amino acid of the 1st PTH cycle. Thevarious peptides are sequenced over 4 to 12 stages. Tables 1 to 7 showthe protein sequences determined.

TABLE 1 Peptide sequences of the substrates Peptides Determined peptidesequences of the substrate 1. Dihydro-lysobactinLeu-Leu-Phe-Leu(OH)-Leu-Arg-Ile- (allo)Thr-Gly-Asn(OH)-Ser 2.Octahydro-lysobactin Leu-Leu-PTHAla(3-cyclohexyl)*-Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser

TABLE 2 Sequence analysis of various peptides (1-3) or peptide fragments(1-3) of the chymotrypsin cleavage Determined peptide sequences of thechymotrypsin Peptides cleavage product 1. Peptide 4-11Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 2. Peptide 1-3 Leu-Leu-Phe3. Peptide 1-3 Leu-Leu-PTHAla(3-cyclohexyl)* *PTHAla(3-cyclohexyl) isnot detectable as a peak with the PTH system used.

TABLE 3 Sequence analysis of various peptides and peptide fragments ofthe subtilisin cleavage of dihydro- and octahydrolysobactin (1-4). Thecleavage product 4-10 is only formed to a greater extent after 24 h.Determined peptide sequences of the subtilisin cleavage Peptidesproduct 1. Peptide 4-11 Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 2.Peptide 4-10 Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 3. Peptide1-3 Leu-Leu-Phe 4. Peptide 1-3 Leu-Leu-PTHAla(3-cyclohexyl)**PTHAla(3-cyclohexyl) is not detectable as a peak with the PTH systemused.

TABLE 4 Sequence analysis of various peptides and peptide fragments ofthe thermolysin cleavage of dihydro- and octahydrolysobactin (1-3).Determined peptide sequences of the thermolysin Peptides cleavageproduct 1. Peptide 3-11Phe-Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 2. Peptide 3-11PTHAla(3-cyclohexyl)*-Leu(OH)-Leu-Arg-Ile-(allo)Thr- Gly-Asn(OH) 3.Peptide 1-2 Leu-Leu *PTHAla(3-cyclohexyl) is not detectable as a peakwith the PTH system used.

TABLE 5 Sequence analysis of various peptides and peptide fragments ofthe papain cleavage of dihydro-(1,2,3,4) and octahydrolysobactin(1,2,3,5). Cleavage product 4-10 is only formed to a greater extentafter 24 h. Determined peptide sequences of the papain cleavage Peptidesproduct 1. Peptide 5-11 Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 2. Peptide5-9 Leu-Arg-Ile-(allo)Thr-Gly 3. Peptide 10-11 Asn(OH)-Ser 4. Peptide1-4 Leu-Leu-Phe-Leu(OH) 5. Peptide 1-4Leu-Leu-PTHAla(3-cyclohexyl)*-Leu(OH) *PTHAla(3-cyclohexyl) is notdetectable as a peak with the PTH system used

TABLE 6 Sequence analysis of various peptides and peptide fragments ofthe proteinase K cleavage of dihydro-(1,2) and octahydrolysobactin(3,4). Determined peptide sequences of the proteinase K Peptidescleavage product 1. Peptide 4-11Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 2. Peptide 5-11Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH)-Ser 4. Peptide 4-10Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH) 5. Peptide 5-10Leu-Arg-Ile-(allo)Thr-Gly-Asn(OH) 6. Peptide 1-2 Leu-Leu

TABLE 7 Sequence analysis of various peptides and peptide fragments ofthe bromelain cleavage of open-chain lysobactin (1,4), dihydro-(2,4) andoctahydrolysobactin (3,4). Determined peptide sequences of the bromelainPeptides cleavage product 2. Peptide 1-9Leu-Leu-Phe-Leu(OH)-Leu-Arg-Ile-(allo)Thr-Gly 3. Peptide 1-9Leu-Leu-PTHAla(3-cyclohexyl)*-Leu(OH)-Leu- Arg-Ile-(allo)Thr-Gly 4.Peptide 10-11 Asn(OH)-Ser *PTHAla(3-cyclohexyl) is not detectable as apeak with the PTH system used.

Example 29 Amino Acid Analysis

Amino acid analysis is an important qualitative and quantitativeparameter for characterizing proteins. In addition to the proteincontent, in the case of known primary structure, the number of theindividual amino acids is determined. The amino acid analysis oflysobactin derivatives and peptide fragments is in good agreement withthe theoretical values from the primary structure (Table 8).Non-proteinogenic amino acids are only quantified in the presence ofcorresponding standards.

100 μg of the lysobactin derivatives and peptide fragments are dissolvedin 200 n1 of 6 N hydrochloric acid and hydrolyzed at 166° C. for 1 h.About 5 nmol of the samples are introduced into the amino acid analyzer.The amount of amino acid is determined via a 4 nmol amino acid standard.

TABLE 8 Amino acid analysis of dihydro-, octahydrolysobactin, dihydro- +octahydrolysobactin, fragment 4-11 and 1-3. The integers are based onIle = 1 or Leu = 2. Dihydro- + Amino Peptide Theoretical PeptideTheoretical octahydro- Dihydro- Octahydro- Theoretical acid 1-3 numbers4-11 numbers lysobactin lysobactin lysobactin numbers Asx(OH) n.d. 1n.d. n.d. n.d. 1 Asx alloTHR 1.04 1 0.91 1.11 1.01 1 Ser 0.59 1 0.890.99 0.90 1 Glx Gly 1.11 1 1.15 1.17 1.12 1 Ala Val Met Ile* 1.00 1 1.001.00 1.00 1 Leu* 2.00 2 1.04 1 2.93 2.33 2.53 3 Tyr Phe 1.01 1 0.55 1.081 Ala(3- n.d. n.d. 1 cyclo- hexyl) Leu(OH) n.d. 1 n.d. n.d. n.d. 1 LysArg 1.06 1 1.05 1.18 1.15 1 Phe(OH) 1 Sum AS 3.01 3 5.83 8 9.39 8.857.71

Example 30 Reverse-Phase Chromatography

In the HPLC chromatography of proteins on chemically bound reversedphases, a bond to the phase used is formed via a hydrophobic interactionof the proteins. The peptides are displaced by organic solvents (mobilephase) according to the strength of their bond to the stationary phase.For this reason, this method is a good criterion for assessing thepurity of a peptide and for monitoring the rate of enzymatic cleavageand the resulting cleavage products. The peptides dihydrolysobactin andoctahydrolysobactin elute from the RP-18 phase at about 35 min and about38 min, fragment 4-11 at about 16 min, 1-3 (LLF) at about 31 min and 1-3(LLA(3-cyclohexyl)) at about 37 min FIG. 1 shows the time course of apreparative enzymatic cleavage with chymotrypsin (Example 11).

About 20 μg of the enzymatic cleavage products and the startingcompounds dihydrolysobactin and octahydrolysobactin or the mixture arechromatographed on a Zorbax 300SB-C18 column (4.6 mm×150 mm; 3.5 μmmaterial; 300 angström pore diameter). The eluent used is anacetonitrile/TFA gradient. Conditions: solvent A 0.1% TFA, solvent B 60%acetonitrile/0.1% TFA; flow 0.7 ml/min, column temperature 40° C., UVdetection 210 nm, solvent A 0.1% TFA, solvent B 0.1% TFA/60%acetonitrile; gradient: 0 min 0% B, 2 min 10% B, 50 min 80% B, 52 min100% B, 55 min 0% B, 60 min 0% B.

Example 31 Capillary Zone Electrophoresis (CZE)

Capillary electrophoresis permits the separation of peptides andproteins on the basis of their charge in an electrical field. Thequality of the separation depends on the buffer, the pH, the temperatureand the additives used. The capillaries used are so-called fused silicacolumns having an internal diameter of 50-100 μm. This method is a verygood criterion for assessing the purity of a peptide and for monitoringthe formation of enzymatic cleavage products. The peptidesdihydrolysobactin and octahydrolysobactin elute from the capillarycolumn at about 21 min, fragment 4-11 at about 18 min, 1-3 (LLF) atabout 24 min, 1-3 (LLA(3-cyclohexyl)) at about 22 min, the deamidatedforms as a double peak at about 30 min (1-11) and 24 min (4-11). FIG. 2shows the time course of an enzymatic cleavage of octahydrolysobactinwith chymotrypsin (Example 5). The great increase in deamidated productsafter 24 h in the buffer can clearly be seen.

About 4 ng of the enzymatic cleavage products or the starting compoundsdihydrolysobactin and octahydrolysobactin, or the mixture, areinvestigated by means of capillary electrophoresis on a glass column(length 72 cm, internal diameter 50 μm). Conditions: current 90 μA,column temperature 25° C., 100 mM phosphate buffer pH 3.0, UV detection210 nm, loading under pressure 3 seconds.

Example 32

Molecular Weight Determined by HPLC-ESI-MS

Peptides and enzymatic cleavage products are separated by RP-18-HPLCchromatography and the molecular weight is determined by electron sprayionization (ESI).

About 100 μg of chymotrypsin cleavage of the mixture ofdihydrolysobactin and octahydrolysobactin are separated with a ZorbaxC18-HPLC column under the following conditions: solvent A 0.1% TFA,solvent B 60% acetonitrile/0.1% TFA; flow 0.7 ml/min, column temperature40° C., UV detection 210 nm, solvent A 0.1% TFA, solvent B 0.1% TFA/60%acetonitrile; gradient: 0 min 0% B, 2 min 10% B, 50 min 80% B, 52 min100% B, 55 min 0% B, 60 min 0% B. The peptides are transferred to theatmospheric pressure ion source of the mass spectrometer and ionizedthere. From there the ions are transferred to the high vacuum region ofthe mass spectrometer and detected. Table 9 shows the molecular weightsdetermined.

TABLE 9 Molecular weights of dihydrolysobactin, octahydrolysobactin andenzymatic cleavage products compared with the theoretical molecularweight (MW) in Dalton. MW Theoretical Peptides in Da MW in Da 1.Dihydrolysobactin 1279 1278.5 2. Octahydrolysobactin 1285 1284.6 3.Peptide 4-11 905 905 4. Peptide 1-3 (LLF) 391 391 5. Peptide 1-3(LLA(3-cyclohexyl)) 397 397 6. Peptide 1-9 1062 1061.5 7. Peptide 1-9(A(3-cyclohexyl)) 1068 1067.6

Example 33 Preparative Chymotrypsin Cleavage of the MixtureDihydro/Octahydrolysobactin

18.27 g of dihydro- and octahydrolysobactin (about 5:4) are dissolved in365 ml of methanol and diluted to 3654 ml with chymotrypsin (731 mg) andcleavage buffer. The reaction is carried out for 30 min at 37° C. andthen stopped with 20 ml of TFA and 150 ml of acetonitrile. Before theaddition of the enzyme, the solutions are warmed to 37° C. in a dryingcabinet. Aliquots of 200 μl are taken after 0 and 0.5 h and the enzymecleavage is stopped with 200 μl of 0.1% TFA in 30% acetonitrile/70%water. The samples are analyzed by HPLC (retention time fragment 4-11about 3.6 min, fragment 1-3 (LLF) about 9.6 min, fragment 1-3(LL(hexahydro)F) about 11.3 min.) (eluent A: 0.1% TFA in water, eluentB: 0.1% TFA in 60% acetonitrile/40% water, gradient: 0 min 30% B, 10 min80% B, 11 min 100% B, 12 min 30% B, 15 min 30% B; flow: 0.7 ml/min,column temperature: 40° C., detection: 210 nm). Alternatively, method 11is used. The solution is divided into 9×500 ml portions and frozen at−70° C. until preparative RP separation. Fragment 4-11 is isolated bypreparative HPLC in several runs.

Preparative separation of fragments 1-3 and 4-11:

About 800 ml of the cleavage solution are filtered through a cartridge(0.2 μm) and chromatographed in two portions of about 400 ml on a Source15RPC column (column size: 2360 ml) using a methanol/TFA gradient.Eluent A: 0.1% TFA in water, eluent B: 0.1% TFA in 100% methanol; flow:30 ml/min; detection 215 nm. The gradient is run according to columnvolumes: after application, the column is washed with 3.6 column volumesof eluent A, and then in 18 column volumes to 45% B, in 0.67 columnvolumes to 100% B, 1.3 column volumes 100% B, in 0.67 to 0% B, 7 columnvolumes of eluent A for equilibration.

10.36 g (77% of theory) of fragment 4-11 are obtained as product.

HPLC/UV-Vis (Method 4): R_(t)=0.5 min

LC-MS (Method 1): R_(t)=10 min;

MS (ESIpos.): m/z (%)=453.6 (100) [M+2H]². 906 (10) [+H]⁺.

MS (ESIneg.): m/z (%)=904 (100) [M−H]⁻.

1. A method for the synthesis of lysobactin derivatives using lysobactinfragment 4-11.
 2. A method for preparing open-chain lysobactinderivatives, whereby lysobactin fragment 4-11 is reacted with atripeptide having a C-terminal aromatic or hydrophobic amino acid in abuffer medium with addition of a C1₋₄-alcohol, whereby the tripeptide ispresent in the form of the free acid or an ester and whereby theconcentration of the C1₋₄-alcohol in the reaction medium is greater than40%.
 3. The method of claim 2, whereby methanol is used as C1₋₄-alcohol.